Discovery and Development of Broad-spectrum Protease Inhibitors of Flaviviruses of Significant Public Health Threats

项目摘要 黄病毒家族的蚊媒成员，包括寨卡病毒(Zkv)、登革病毒(DNV)和西尼罗河病毒 由于最近流行的频率和严重性，病毒(WNV)被归类为重新出现的病原体。也称为 作为虫媒病毒，这些病毒是许多影响人类人口的衰弱疾病的病原体。 全世界。因此，媒介传播的疾病现在占全世界所有感染的17%。DNV是最快的 日益严重的虫媒病毒病目前每年影响4亿人，其中9600万人表现为临床严重 22000人死亡，主要是儿童。西尼罗河病毒被认为是世界范围内病毒性脑炎的最重要病原体。 最近爆发的寨卡病毒感染与先天性小头畸形和颅内钙化有关，在 成人，患有GBS和严重的血小板减少症。目前还没有有效的治疗方法来治疗由这些疾病引起的感染 病毒，这突出表明迫切需要找到预防和治疗措施。 黄病毒科的基因组被翻译成一个多聚蛋白，加工后产生3个结构蛋白和7个结构蛋白 非结构蛋白。多聚蛋白的正确处理对于所有黄病毒的复制是必不可少的，这需要 宿主蛋白酶和高度保守的病毒NS2B-NS3蛋白酶(NS2B-NS3pro)。因此，病毒蛋白酶是一种 开发阻止黄病毒复制的小分子抑制剂的合理目标。小分子抗病毒药物 针对HIV-1编码的和编码的丙型肝炎病毒编码的蛋白酶已经成功地被开发出来，这支持了 开发针对黄病毒NS2B-NS3Pro的化疗药物。我们建议的创新之处在于：(一) 用于鉴定低结合片段HITS的高灵敏度筛选方法的优化；(Ii)进化 片段和化合物进入广谱导联；(Iii)使用复制子和斑块分析来测试细胞效力 引导优化。我们的初步结果和多个基于细胞的模型的使用支持了 广谱抗黄病毒疗法的发现。具体目标是：目标1：完成片段的筛选 以及用于鉴定广谱NS2B-NS3Pro HITS的化合物文库。里程碑1：从结构上确定6-8个 独特的广谱NS2B-NS3PRO与IC50≤25μM.目标2a：迭代3D结构和基于合成孔径雷达的发现 三个不重叠的广谱NS2B-NS3Pro抑制剂系列，使用以下组合：(I)商业类似物 (2)医学化学设计与综合方法。里程碑2：确定三个不重叠的广谱NS2B- NS3Pro缓蚀剂系列，IC50≤200 nm。目标2b：表征生化有效的抑制剂：(A)模式 抑制和(B)酶：与IC50≤200 nm的配基相互作用和优先化合物，用于细胞功效研究。 目的3a：体外评价和优化具有生物化学活性的化合物的细胞效率(EC50)和 细胞毒性(CC50)。里程碑3：使用EC50≤5μM和CC50≥200μM.从每个系列中确定4-6个先导化合物 3B：对具有广谱体外活性和可接受的化合物进行基于ADME的体外铅优化 治疗指标。第一阶段里程碑：确定2-4种具有高生物利用度、弱抑制作用的先导化合物 CYP450稳定性最佳，不是HERG通道阻滞剂。

Project Summary Mosquito-borne members of the Flavivirus family including Zika virus (ZKV), Dengue virus (DNV) and West Nile virus (WNV), are classified as re-emerging pathogens due to the frequency and severity of recent epidemics. Also known as arboviruses, these viruses are the etiologic agents of many debilitating diseases affecting the human population worldwide. Consequently, vector borne diseases now account for 17% of all infections worldwide. DNV is the fastest growing arboviral disease currently affecting 400 million annually with 96 million cases manifesting into clinical severity and 22,000 deaths, mainly children. WNV is considered the most important causative agent of viral encephalitis worldwide. The recent ZKV infection outbreak has been associated with congenital microcephaly and intracranial calcification and, in adults, with GBS and severe thrombocytopenia. Currently there is no effective treatment for infections caused by these viruses, which highlights the urgent need to find preventive and therapeutic interventions. The Flaviviridae genome is translated into a single polyprotein which is processed to yield 3 structural and 7 nonstructural proteins. The correct processing of the polyprotein is essential for replication of all flaviviruses, which requires both host proteases and the highly conserved viral NS2B-NS3 protease (NS2B-NS3pro). Hence, the viral protease is a rational target for development of small molecule inhibitors that block flavivirus replication. Small molecule antivirals targeting HIV-1-encoded and HCV-encoded proteases have been successfully developed, which supports the concept of developing chemotherapeutic agents targeting the flavivirus NS2B-NS3pro. The innovation of our proposal is: (i) optimization of a highly-sensitive screening assay for the identification of low binding fragment hits; (ii) evolution of fragment and compound hits into broad-spectrum leads; (iii) the use of replicon and plaque assays to test for cellular efficacy and guide optimization. Our preliminary results and the use of multiple cell-based models supports the feasibility of the discovery of broad-spectrum anti-flaviviral therapeutics. The specific aims are: Aim 1: Complete the screening of fragment and compound libraries for the identification of broad-spectrum NS2B-NS3pro hits. Milestone 1: Identify 6-8 structurally distinct broad-spectrum NS2B-NS3pro hits with an IC50 ≤ 25μM. Aim 2a: Iterative 3D-structure and SAR-based discovery of three non-overlapping broad-spectrum NS2B-NS3pro inhibitor series, using a combination of: (i) commercial analogues and (ii) med-chem design and synthesis approach. Milestone 2: Identify three non-overlapping broad-spectrum NS2B- NS3pro inhibitor series with an IC50 ≤ 200nM. Aim 2b: Characterize biochemically potent inhibitors for: (a) mode of inhibition and (b) enzyme:ligand interactions and prioritize compounds with IC50 ≤ 200nM, for cellular efficacy studies. Aim 3a: In vitro evaluation and optimization of biochemically potent compounds for cellular efficacy (EC50) and cytotoxicity (CC50). Milestone 3: Identify 4-6 lead compounds from each series with EC50 ≤ 5μM and CC50 ≥ 200μM. Aim 3b: Conduct in vitro ADME-based lead optimization of compounds with broad spectrum in vitro activity and acceptable therapeutic indices. Phase I Milestone: Identify 2-4 lead compounds exhibiting high bioavailability, weak inhibitor of CYPP450s, optimum stability and are not hERG channel blockers.